In 1967, during research involving the purification of DNA-dependent RNA polymerase from homogenate of maize seedlings, two ATP incorporating activities were resolved by chromatography [Stout et al., Biochem. Biophys, Acta. 134, 327 (1967)]. The major activity was identified as containing RNA polymerase. The second enzyme-containing activity catalyzes the sequential addition of AMP (adenylic acid) moieties from ATP (adenosine triphosphate) to the 3'-hydroxyl terminus of ribo- or deoxyribopolymers [Walter et al., Biochem Biophys, Acta. 217, 72-82 (1970); Mans et al., Biochem. Biophys. Acta. 247, 113-121 (1971); Mans, Biochem. and Biophys. Res. Comm., 45, 980-983 (1971); Mans et al., J. Biol. Chem., 250, 3672-78 (1975); Walter et al., Plant Physiol., 56, 821-25 (1975)].
The unique ability of the activity to utilize both ribo- and deoxyribopolymers as acceptor molecules for polyadenylylation render the enzyme commerically desirable as a reagent for the radioactive "tailing", for example, of RNA and DNA molecules in programs and systems involving the molecular cloning of specific viral prokaryotic and eukaryotic gene sequences. The enzyme containing activity isolated from mammalian cells does not have the ability to utilize deoxyoligomers as primer [Mans et al., Life Sciences, 14, 437-445 (1974)].
The disclosures of each of the references cited hereinabove are incorporated herein by reference.
The utilization of the heretofore only partially purified activity suffered from several disadvantages. The presence of significant exonuclease activity in the partially purified preparations limits the length of the poly A sequence of the accumulated product. The presence of traces of endonucleolytic activities randomly cleave high molecular weight RNA primers, resulting in the formation of less than full-length cDNA probes. Significant DNA endonucleolytic activity introduces random single stranded nicks in polydeoxribonucleotide primers resulting in less than full-length, labeled restriction endonuclease fragments. The activity has significant but relatively low activity on deoxyoligomers as compared with the polyadenylylation of RNA primers of comparable length. The enzyme is labile on storage at 4.degree. C. and upon successive freezing and thawing.
It is an object of the present invention to provide in substantially pure form, the ATP; polynucleotide adenylyltransferase enzyme and a method for its isolation and purification.